Discovery of COX-2 and 5-LOX dual targeted inhibitors from Nauclea officinalis by employing a combination of affinity ultrafiltration and HPLC-MS/MS

Nauclea officinalis (N. officinalis), a medicinal plant of the genus Nauclea in the Rubiaceae family, is used to treat bronchitis, pneumonia and acute tonsillitis and enteritis in China. Extracts of N. officinalis include more than 60 alkaloids, which exert all kinds of pharmacological effects, such as antiproliferative, antiparasitic, antimicrobial, anticancer, and anti-inflammatory activities. In this study, an affinity ultrafiltration and high-performance liquid chromatography- tandem mass spectrometry (AUF-LC-MS/MS) approach was constructed and validated, and was applied to rapidly screen COX-2/5-LOX inhibitors in N. officinalis extracts (NOE). To gain a more comprehensive and profound understanding of the anti-inflammatory mechanism of N. officinalis. Central Composite Design (CCD) of Response Surface Method (RSM) was used to optimize extraction conditions for potential COX-2/5-LOX inhibitors from NOE. Next, COX-2/5-LOX dual targeted inhibitors were screened from NOE using AUF-LC-MS/ MS, and the biological activity of the screened ligands was demonstrated by enzyme inhibition assay. Finally, the preferred binding modes for these COX-2/5-LOX inhibitors were then determined by molecular docking, and compared with active drugs celecoxib and zileuton, respectively, to further explore ligand active binding sites and intersections between ligands and target proteins. The optimum extraction conditions were follows: ethanol concentration of 79 %, liquid - solid ratio of 11.8 v/w and extraction time of 1.8 h. The results of COX-2 inhibition assay showed that angustine (AU) had the more stronger inhibition activity with an IC50 of 20.49 mu M, followed with IC50 of angustidine (AD) and nauclefine (NF) were 35.89 and 60.45 mu M, respectively. Among the three components, AU also exhibited the highest inhibition of 5-LOX with an IC50 of 1.18 mu M, followed by NF and AD at 2.75 and 4.35 mu M, respectively. Owing to the synergistic effect of multiple components make NOE exhibit strong inhibitory activity, it is shown potent inhibitory activities with an IC50 at 9.47 and 6.16 mu g/mL for COX-2 and 5-LOX, respectively. This work has demonstrated that NF, AD, and AU are potential COX-2/5-LOX dualtarget inhibitors, indicating that N. officinalis anti-inflammatory activities were partially based on these COX-2/5LOX inhibitors. These research results can also be further used for developed or improved anti-inflammatory agents with more beneficial effects.